Fig. 3: Identification of AR-regulated lncRNAs.

a Schematic depicting the analysis to identify lncRNAs upregulated in CRPC tissues. We checked whether obtained lncRNAs in Fig. 1 (Type_A oe Type_B) are truly lncRNA or not by viewing genome browser. Of note, we excluded lncRNAs that are composed of exons of protein-coding genes because they could not be distinguished from protein-coding genes by RNA-seq analysis. A total of 91 Type_A and 72 Type_B candidate lncRNAs were identified. b Classification of lncRNAs identified in the present study. LncRNAs were classified to antisense RNA, which is situated at the antisense region of protein-coding genes, intergenic lncRNA, which is not overlapped with protein-coding gene, and others (sense, overlapping, or miRNA precursor). PCAT1 is shown as a representative lncRNA upregulated in CRPC. P value was determined by Mann–Whitney U test. c Expression levels of representative AR-regulated lncRNAs (HOTAIR, ARLNC1, CTBP1-AS, PCGEM1) in RNA-seq analysis. P value was determined by Mann–Whitney U test. N.S. not significant. d Flow chart of the investigation of androgen-regulated lncRNAs upregulated in CRPC tissues. We used RNA-seq in 22Rv1 to obtain AR-repressed (fold>1.5 by siAR) or AR-induced (fold<0.8 by siAR) lncRNAs. We also obtained lncRNAs repressed by androgen (fold<0.8) or induced by androgen (fold>1.5). See also Supplementary Fig. 3. Then we selected 21 transcripts with AR binding in the vicinity (within 10 kb from TSSs) for further studies (Fig. 4a). e Representative lncRNAs upregulated in CRPC. RNA-seq results of CRPC-Lnc #6 (PRKAG2-AS1) and CRPC-Lnc #9 (HOXC-AS1) are shown. ChIP-seq signals, AR-binding signals with vehicle or DHT treatment, and active histone modification signals (AcH3, K4me3) in 22Rv1 cells relative to input control are also shown. In HOXC locus, other lncRNAs, HOXC-intron and HOTAIR, were also upregulated in CRPC.